African trypanosomiasis (sleeping sickness) is a parasitic infection that is fatal if not treated. The incidence of disease is rising, there are no vaccines, and current therapy is inadequate. This is an ongoing project to study DNA topoisomerases as potential targets for rational new antitrypanosomal drug therapy. The topoisomerases are enzymes essential for the orderly synthesis of nucleic acids and for cell survival, furthermore, they are proven targets for a variety of clinically important antibacterial and antitumor drugs. Previous work from this laboratory has shown that potent and specific inhibitors yield valuable insights into the normal function of topoisomerases in trypanosomes and that the severity of the resulting lesions correlates with cytotoxicity. Recent studies have revealed that trypanosomes have a highly unusual topoisomerase I gene that lacks the expected active site motif. The specific aims of the proposed work are three: First is to study topoisomerase I: characterize the enzyme itself (identify the active site residue, obtain an x-ray crystal structure) and define the metabolic consequences of topo I inhibition. The second aim is to continue studies of topoisomerase II by isolating a nuclear enzyme and evaluating the effects of topo II inhibitors on nuclear DNA metabolism. The third goal is to bring these molecular studies closer to the clinic by assessing the structure-activity relationship of topoisomerase inhibitors, seeking compounds that are selectively toxic for parasites, compared with mammalian cells. Inhibitors that appear most promising will be evaluated in mice. This work will increase our understanding of these critical enzymes in trypanosomal metabolism, and it offers some hope for the development of much-needed new therapy.